Genetic characterization and fine mapping BrCER4 in involved cuticular wax formation in purple cai-tai (Brassica rapa L. var. purpurea)

Plant aerial organs are covered by waxy cuticles, which are complex mixtures of very long-chain fatty acids and their derivatives. This waxy cuticle can reduce uncontrolled water loss and environmental damage, but a lack of waxy cuticle generates a glossy phenotype, a favorable trait for many crops. The mechanism underlying the glossy phenotype of purple cai-tai (Brassica rapa L. var. purpurea), a unique stem vegetable, has not yet been studied. Genetic analysis indicated that the waxy cuticle in purple cai-tai is controlled by a single locus located on chromosome A01. The trait was ultimately mapped between two markers that are approximately 310kb apart. Seventy-five genes were identified in the target region. Based on gene annotation, two genes in this region, Bra011487 and Bra011470, are known to be involved in the cuticular wax biosynthesis pathway. No sequence variation for the Bra011487 gene was found between the two parents, while Bra011470 had a 39bp deletion in the glossy parent. Bra011470 (BrCER4) is a homolog of CER4 (ECERIFERUM4) in Arabidopsis. It was expressed in the leaves, stems, flowers and siliques, and its expression was decreased in the glossy mutant. Analysis with transmission electron microscopy revealed that the cuticular ultrastructure of the mutant leaves and stem was dramatically altered compared with that of the wild-type leaves and stem. Analysis of the cuticular wax of mutant stems further confirmed a severe decrease in primary alcohols. These results indicated that BrCER4 is responsible for surface wax biosynthesis and that a loss-of-function mutation in this gene results in the glossy phenotype. A genetic marker flanking the potentially causal deletion was developed and may be useful for marker-assisted selection in future breeding programs for B. rapa.